There is an urgent need for alternatives to traditional antibiotics for the treatment of bacterial infections. The small number of novel antibiotics developed over the past 20 years combined with the misuse/application of antibiotics has led to increased frequencies of bacterial pathogen resistance to all classes of antibiotics. In both procaryotic and eucaryotic cells, examples of antisense mechanisms have been found that lead to inhibition of transcription and translation and in turn lead to inhibition of cell growth. In eukaryotic cells, the short interference RNA-DNA hybrid molecules (siHybrids) have been shown to inhibit both transcription and translation of genes and hold promise as antisense therapies. In bacteria, the small regulatory RNAs (srRNAs) have been shown to base pair with target mRNAs and affect their translation and stability. The peptide nucleic acids (PNAs) are very stable, short synthetic nucleic acid analogues in which the sugar-phosphate backbone of natural nucleic acid has been replaced by a synthetic peptide backbone usually formed from N-(2-aminoethyl)glycine units. PNAs are very long-lived antisense constructs that inhibit expression of targeted genes and can inhibit growth in a number of bacterial species. However, the effectiveness of the PNAs against intracellular bacterial pathogens has not been well characterized. The proposed study will investigate the use of PNAs alone or combined with immunoliposomal nanoplex approaches in inhibiting gene expression in the intracellular bacterial pathogen Brucella. Brucellosis in humans, principally caused by one of three Brucella spp., is one of the five most common bacterial zoonoses in the world. Moreover, the disease is very difficult to treat with available antibiotics with a relapse rate of up to 15%. The hypothesis is that PNAs can be used to inhibit gene expression in Brucella both in culture as well as in infected macrophages. Aim-1: Identify additional PNAs and determine the MICs of specific PNAs that inhibit growth of B. suis. Aim-2: Assess whether immunoliposomal nanoplexes can target-delivery of PNAs to B. suis infected macrophages and inhibit pathogen growth. PUBLIC HEALTH RELEVANCE: There is an urgent need for alternative therapeutics to treat bacterial infections. Anti-sense gene therapeutics has been shown to kill bacterial pathogens in pure culture but not efficiently in infected tissues. What is missing is a delivery system that will target the anti-sense genes to the pathogen particularly when it is living inside of infected tissues. The proposed study will investigate the use of targeting approaches to enhance delivery of anti-sense genes into infected tissues to kill the bacterial pathogen.